While adoptive T-cell therapy has yielded instances of dramatic clinical success in treating refractory diseases, many patient responses remain partial, particularly in the treatment of solid tumors. The goal of this research is to improve the efficacy of adoptive T-cell therapies by addressing two interrelated challenges: countering the solid-tumor microenvironment's immunosuppressive effects on T cells, and supporting robust, persistent, tumor-localized anti-cancer functions by transferred T cells. Overproduction of transforming growth factor beta (TGF-) by tumor cells inhibits cytotoxic T-cell functions and promotes immunosuppressive regulatory T-cell development and activity in the tumor microenvironment. We hypothesize that high local TGF- concentrations can serve as a tumor microenvironment marker and that a synthetic, TGF--responsive transcription system can be programmed to both reduce TGF-'s endogenous immunosuppressive effects and induce robust anti- tumor responses. These hypotheses will be tested through three specific aims. In Specific Aim 1, a synthetic TGF--inducible transcription system will be constructed that can simultaneously compete against the endogenous immunosuppressive signaling pathway and link the presence of TGF- to the production of a customizable transcriptional output. In Specific Aim 2, a variety of candidate genes will be integrated into the synthetic transcription system to couple the presence of TGF- to the promotion of cell-intrinsic T-cell activities, including strengthening T-cell activation, improving T-cell proliferation, and/or enhancing T-cell cytotoxicity. In Specific Aim 3, the synthetic transcription system will be coupled to genetic outputs that recruit native immune components and modify the tumor microenvironment. The proposed genetically encoded TGF--inducible transcription systems will be constructed using isothermal DNA assembly and standard molecular cloning techniques. Synthetic transcription systems coupled to functional genetic outputs will be integrated into established and primary human T cells for in vitro evaluation of proper expression and anti-tumor activities using quantitative real-time PCR, western blots, surface and intracellular antibody staining, and effector/target cell co-culture assays that quantify the effects of engineered T cells on target cel lysis, cell-cycle arrest, and migration. Western blots and intracellular antibody staining will als be used to evaluate the synthetic transcription system's inhibitory effects on endogenous TGF- signaling. Systems with the most promising genetic outputs and robust inhibitory effects on endogenous TGF- signaling activities will be integrated into the pmel-1 mouse model for adoptive T-cell therapy to evaluate in vivo anti-tumor T-cell functions and improvements in therapeutic efficacy. This research aims to address a critical barrier to progress in T-cell therap for cancer by pursuing the de novo construction of multi-functional genetic constructs previously unavailable in the T-cell therapy toolbox, thereby generating T cells with more robust and precisely targeted anti-tumor activities for immunotherapy against cancer.